In general, the invention relates to methods of labeling proteins.
Covalent conjugates of polypeptides with non-peptide xe2x80x9clabelsxe2x80x9d or xe2x80x9ctagsxe2x80x9d form a useful class of reagents in protein research. A conjugate is usually intended to retain the native properties of the protein while gaining a new, non-native property due to the label. Biotinylation, for example, permits proteins to be separated, quantified, or immobilized by mechanisms based on the strong interaction of biotin with avidin or streptavidin (Bayer and Wilchek (1990) Protein Biotinylation, Meth. Enzymol. 184:138-160). Fluorescent or metal-chelating groups can also be introduced to generate newly modified proteins.
It would be convenient to be able to introduce a non-protein label as the protein is produced, but this is only sometimes feasible, for example, if the peptide can be produced by chemical synthesis, and is impractical when the protein is produced biologically. Generally, the conjugate must be formed by treating the peptide with a functional group-specific reagent that contains the label. Moreover, unless the peptide contains only one group attacked by the reagent, this procedure generally yields a mixture of products. This random form of labeling is sometimes adequate, but it is often preferable to modify a protein at a single specified site and to employ the modified product in purified form. For such cases, it would be valuable to have a method of directing the modifying group to a single, preselected location. Such a precisely targeted modification is termed site-directed protein tagging.
In general, the invention features a protein having a covalently bonded puromycin-tag, the tag being positioned at the C-terminal end of the protein.
In preferred embodiments, the tag is a small molecule (for example, biotin); the tag is a detectable label (for example, fluorescein, rhodamine, or BODIPY, or derivatives thereof); the tag is a functional group (for example, a functional group having a reactivity orthogonal to the reactivity of one of the protein""s functional groups); the tag is a tether for attachment to a solid support (for example, a column, bead, or chip); the tag is one member of a specific binding pair; the tag is a phenyl diboronic acid derivative; the puromycin-tag further includes a nucleotide sequence positioned between the tag and the puromycin; and the nucleotide sequence is between about 1-200 nucleotides in length.
In a related aspect, the invention features a method for C-terminal protein tagging, involving (a) providing a nucleic acid sequence encoding the protein; (b) translating the sequence under conditions in which translation stalls at the 3xe2x80x2 end of the sequence, forming a stalled translation complex; and (c) contacting the stalled translation complex with a puromycin-tag under conditions in which the puromycin-tag is covalently bonded to the C-terminus of the protein.
In preferred embodiments, the tag is attached to the 5xe2x80x2-hydroxy group of puromycin; the tag is attached to the 5xe2x80x2-hydroxy group of the puromycin through a phosphate group; the nucleic acid sequence encoding the protein contains no stop codons; the translation step is carried out in the substantial absence of at least one translation release factor; the 3xe2x80x2-end of the nucleic acid sequence encoding the protein is covalently linked to a DNA oligomer; the tag is a small molecule (for example, biotin); the tag is a detectable label (for example, fluorescein, rhodamine, or BODIPY, or a derivative thereof); the tag is a functional group; the protein has a first functional group and the tag is a second functional group, wherein the first functional group has a reactivity orthogonal to the reactivity of the second functional group; the tag is a tether for attachment to a solid support (for example, a column, bead, or chip); the tag is one member of a specific binding pair; the tag is a phenyl diboronic acid derivative; the puromycin-tag further includes a nucleotide sequence positioned between the tag and the puromycin; and the nucleotide sequence is between about 1-200 nucleotides in length.
By a xe2x80x9cproteinxe2x80x9d is meant any two or more naturally occurring or modified amino acids joined by one or more peptide bonds. xe2x80x9cProtein,xe2x80x9d xe2x80x9cpeptide,xe2x80x9d and xe2x80x9cpolypeptidexe2x80x9d are used interchangeably herein.
By a xe2x80x9cpuromycin-tagxe2x80x9d is meant puromycin having a covalently bonded structural or functional moiety which is not native to the puromycin molecule and which is chosen from the group consisting of a detectable label, a chemically reactive functional group, a small molecule, a protein or peptide, a peptoid, a naturally occurring or non-naturally occurring polymer, a solid-phase bound tether, a carbohydrate, or a nucleic acid (preferably, of between about 1-200 nucleotides) which does not encode the protein to which the puromycin-tag is itself covalently linked. By a xe2x80x9cnucleic acidxe2x80x9d is meant any two or more covalently bonded, naturally occurring or modified nucleotides and includes DNA, RNA, and PNA. Preferred puromycin-nucleic acid tags include 5xe2x80x2-C-C-puromycin-3xe2x80x2.
By a xe2x80x9csmall moleculexe2x80x9d is meant a molecule having a molecular weight of approximately 2000 Daltons or less, preferably, 1500 Daltons or less, more preferably, 1000 Daltons or less, and, most preferably, 500 Daltons or less.
By a xe2x80x9cfunctional groupxe2x80x9d is meant any moiety of, or arrangement of atoms in, a molecule which exhibit some chemical reactivity.
The present invention provides a number of advantages over current chemical and enzymatic protein-tagging methods. For example, the tag is introduced in the final step of translation on the ribosome. This modification is advantageous because tagged proteins may be generated in a single preparative step. In addition, the tag is introduced in translation buffer under conditions which enhance protein stability. Again, this provides for increased product yield and optimized protein quality. In particular, although several schemes for N-terminal (Drijfhout et al. (1990) Anal. Biochem. 187: 349-354; Wetzel et al. (1990) Bioconjugate Chem. 1: 114-122) and C-terminal (Schwarz et al. (1990) Meth. Enzymol. 184: 160-162; Rose et al. (1989) Peptides 1988 (G. Jung and E. Bayer, eds.) pp. 274-276, Walter de Gruyter and Co., New York) tagging have been described, many of these methods involve a tagging step that is carried out under conditions which disrupt protein structure. For example, modification at non-physiological pH and temperature or in the presence of non-aqueous solvents or chemicals, followed by purification of the modified protein, disrupts folding thus leading to a non-functional product. In contrast, the present tagging method is performed under native conditions. Finally, in yet another advantage, the present invention enables the introduction of a tag regioselectively at the C-terminus of a protein, facilitating the production of native proteins carrying desired C-terminal structural or functional elements in a simple and efficient way.
The C-terminally tagged polypeptides and proteins produced by the methods of the present invention may be used in any appropriate technique, for example, in any affinity purification method, protein detection method (for example, using proteins having C-terminal fluorescein tags), structure function or protein dynamics analyses (for example, using proteins having C-terminal reporter tags), pharmaceutical analyses (for example, using proteins having detectable C-terminal tags which allow for a determination of cellular protein uptake or cellular localization), or protein display technology (for example, using solid phase tags to generate protein arrays on microchips).
Other features and advantages will be apparent from the following detailed description and from the claims.